A fast and effective method for purification of elongation factor 1 alpha from rabbit liver.
Fast, efficient and gentle method of elongation factor 1 alpha (EF-1 alpha) purification from rabbit liver has been developed. Isolation procedure consists of three steps: gel-filtration of postmithohondrial supernatant on Sephacryl S-400, anion(DEAE-Cellulose)- and cation(SP-Sepharose)- exchange chromatographies. The procedure takes two-three days. Minimal number of the purification steps and the only one buffer usage during entire purification provide the high speed and efficiency of the method. Purity of EF-1 alpha preparation obtained was more than 90% as judged from DS-Na polyacrylamide gel electrophoresis. EF-1 alpha was demonstrated to be highly active in three different assays: the [3H]GDP-binding, the stimulation of [14C]phenylalanyl-tRNA binding to poly(U)-programmed 80S ribosomes and the stimulation of poly[14C]phenylalanine synthesis on poly(U)-programmed 80S ribosomes in the presence of EF-2. Since EF-1 alpha may exist in a cell as either GDP- or GTP-bound form the nature of nucleotide bound was studied using high pressure liquid chromatography (HPLC) analysis. Each molecule of EF-1 alpha appears to contain one molecule of GDP.
Shalak
V F
VF
Budkevich
T V
TV
Negrutskił
B S
BS
El'skaia
A V
AV
D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D002850:Chromatography, Gel / Q000379:methods; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D006153:Guanosine Diphosphate / Q000032:analysis / Q000737:chemistry; D008099:Liver / Q000378:metabolism*; D020648:Peptide Elongation Factor 1; D010445:Peptide Elongation Factors / Q000737:chemistry / Q000302:isolation & purification*; D011072:Poly U / Q000494:pharmacology; D012360:RNA, Transfer, Phe / Q000737:chemistry; D011817:Rabbits; D012261:Ribonucleoproteins / Q000737:chemistry / Q000302:isolation & purification*; D012269:Ribosomal Proteins / Q000737:chemistry
D020648:Peptide Elongation Factor 1; D010445:Peptide Elongation Factors; D012360:RNA, Transfer, Phe; D012261:Ribonucleoproteins; D012269:Ribosomal Proteins; C039913:ribosomal protein S80; D006153:Guanosine Diphosphate; D011072:Poly U